Hypoxia inducible factor (HIF) is a transcriptional complex that plays a key role in mammalian oxygen homeostasis and regulates angiogenic genes such as VEGF. The subunit components, HIF-1 alpha and HIF-1 beta (ARNT), are constitutively expressed and regulation is achieved by the selective destruction of HIF-1 alpha. HIF-1 alpha is a regulatory point of cellular response to hypoxia.
In the presence of oxygen, posttranslational modification by prolyl hydroxylation in the oxygen-dependent degradation domain (ODD) targets HIF-1 alpha subunits for proteasomal degradation via binding to the VHL (von Hippel-Lindau tumor suppressor protein), Elongin C/B, Cul2, Rbx1 ubiquitin E3 ligase complex. However, during ischemia, the hydroxylation of HIF-1 alpha is inhibited and HIF-1 alpha binds to ARNT to form a functional transcriptional activator that turns on genes with hypoxic response elements (e.g. VEGF, EPO, glycolytic enzymes). Proline hydroxylation of HIF-1 alpha is a required step for ubiquitinylation by the E3 ligase complex and is accomplished by three recently described enzymes, EGLN1, EGLN2, and/or EGLN3.
EGLNs are HIF-specific enzymes, distinct from procollagen prolyl-4-hydroxylases (P4H), which are responsible for the formation and stabilization of the triple helical domains in proteins, such as procollagen. EGLNs are human homologs of Caenorhabditis elegans Egl9 prolyl hydroxylase, and have been identified as HIF prolyl hydroxylases. EGLNs require molecular oxygen, iron, and oxoglutarate for activity. Furthermore, their activities are modulated by graded hypoxia and iron chelation and are inhibited by the prolyl hydroxylase inhibitors. These enzymes are distinguished by the peptide motif recognized for hydroxylation, Leu-X-X-Leu-Ala-Pro.
HIF prolyl hydroxylases belong to a family of non-heme iron Fe(II)-dependent oxygenases. HIF prolyl hydroxylases further differentiate into a class of dioxygenases with a requirement for 2-oxoglutarate (2-OG). Structural and mechanistic studies suggest that in the presence of dioxygen a catalytic process occurs in which a Fe:O species is generated during oxidative decarboxylation of 2-OG to succinate. This step is coupled to the oxidation of the substrate, which in the case of HIF, is a proline residue. The reaction results in the generation of succinate and CO2 with oxygen incorporated into the hydroxyl group. Fe(II) is coordinated into the catalytic site of the dioxygenases and 2-OG is ligated to the iron. Iron may be displaced or replaced by other metals such as cobalt ions and render the enzyme inactive. However, further studies into understanding the mechanisms of action have been hampered by the lack of three-dimensional coordinates of EGLNs. The availability of such spatial coordinates may be useful in designing novel ligands or identifying other ligands of EGLNs, which may, in turn, be regulators of HIF-1 alpha-prolyl hydroxylation, which may, in turn, be useful in the treatment of HIF-regulated disorders.